HCN channels play a critical physiological role in many tissues including the brain and heart. These channels are responsible for pacemaker activity in both cardiac and neuronal cells, dendritic integration, and setting resting membrane potentials1. HCN channels have also been implicated in many pathophysiological conditions including epilepsy, peripheral neuropathic pain and Parkinson's disease2-5. In 2004, an accessory protein of HCN channels, termed Trip8b, was discovered by Santora and colleagues6. More recently, three simultaneous studies were published that showed that Trip8b was highly alternatively spliced and that the variants had different effects on trafficking HCN channels to the cell surface7- 9. In addition, these groups showed that all of the variants studied were able to blunt the effect of cAMP on the channel. Gating of HCN channels is regulated by cAMP in a direct, protein kinase or phosphorylation, independent manner, but in the presence of Trip8b that regulation is greatly reduced. In neurons, little is known about the diversity of expression and function of HCN channels. It is known that the trafficking and gating of the channel is different in neurons than in expression systems. HCN channels show a highly specified pattern of expression in neurons, for example, in CA1 pyramidal neurons HCN channels are expressed in a gradient of increasing density with increasing distance from the soma. Given its diversity of function in vivo, Trip8b is an attractive candidate for regulation of HCN channels in vivo. With that in mind, I plan to study the biophysics of the interaction of HCN2 channels and Trip8b. What residues are critical for the interaction for these two proteins? There is increasing evidence that there is a second interaction site in the cyclic nucleotide binding domain (CNBD)7, 10. Are both of these interactions important for the physiological role of Trip8b? What is the stoichiometry of that complex? How does the Trip8b/HCN interaction alter the cyclic nucleotide dependence of channel gating? I plan to address these questions using a combination of fluorescence and electrophysiology that will include patch clamp fluorometry, single molecule fluorescence, and targeted mutagenesis based on crystal structures. In addition to the information provided about Trip8b, I believe over the long term this study will help elucidate the important structures and rearrangements that occur during "normal" HCN channel gating and ligand binding. Also, these finding will be of general interest towards the understanding of gating and ligand binding movements for many different types of ion channel and receptors. PUBLIC HEALTH RELEVANCE: The long term goal of this project is to understand the structural mechanisms of the regulation of HCN channels by the newly discovered accessory subunit Trip8b by applying a combination of structural and functional assays. HCN channels are responsible for many physiologically important functions including pacemaking activity in the brain and heart, modulation of synaptic transmission, and fine tuning the electrical signals in dendrites. Given Trip8b's potentially critical physiological role, elucidating this interaction will be instrumental in understanding HCN channel function in healthy individuals and its misregulation in diseases such as epilepsy and Parkinson's disease.